Silicone surfactants for emulsion assays

ABSTRACT

System, including methods and compositions, for making and using emulsions that include a silicone oil and a silicone surfactant. The emulsions may include aqueous droplets disposed in a continuous phase that includes a silicone oil and a silicone surfactant. The aqueous droplets may contain an analyte, optionally at partial occupancy, and/or a luminescent (e.g., photoluminescent) reporter. An assay of the analyte may be performed with the droplets. In some cases, signals may be detected from the droplets, and a characteristic of the analyte, such as an analyte level or activity, may be determined based on the signals.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/216,006, filed Mar. 17, 2014, and now U.S. Pat. No. 9,758,535, which,in turn, is based upon and claims the benefit under 35 U.S.C. § 119(e)of U.S. Provisional Patent Application Ser. No. 61/789,676, filed Mar.15, 2013. Each of these priority applications is incorporated herein byreference in its entirety for all purposes.

CROSS-REFERENCES TO OTHER MATERIALS

This application incorporates by reference in their entireties for allpurposes the following materials: U.S. Pat. No. 7,041,481, issued May 9,2006; U.S. Patent Application Publication No. 2010/0173394 A1, publishedJul. 8, 2010; U.S. Patent Application Publication No. 2011/0217712 A1,published Sep. 8, 2011; and Joseph R. Lakowicz, PRINCIPLES OFFLUORESCENCE SPECTROSCOPY (2^(nd) Ed. 1999).

INTRODUCTION

An emulsion is a mixture of two or more liquids that are normallyimmiscible (nonmixable or unblendable). Typically, one liquid, referredto as the dispersed phase, is dispersed into the other liquid, referredto as the continuous phase. Emulsions hold substantial promise forrevolutionizing high-throughput assays, as emulsification techniques cancreate thousands, millions, or even billions of discrete aqueousdroplets from a single sample. The resulting aqueous droplets, due totheir isolation from each other within an immiscible continuous phase,can function as independent reaction chambers for biochemical reactions.Significantly, even a small aqueous sample can be partitioned into avast number of droplets. For example, an aqueous sample with a volume of200 microliters can be dispersed into approximately four milliondroplets, each having a volume of 50 picoliters. In this way, individualbiological components (e.g., cells, nucleic acids, proteins, etc.) canbe manipulated, processed, and studied discretely in a massivelyhigh-throughput manner.

Emulsions for assays are often formulated to have a continuous phasethat includes a perfluorinated oil and a perfluorinated surfactant. Theuse of such a fluorophilic continuous phase around droplets can providea permissive surrounding environment for certain biochemical reactions,such as PCR amplification, to occur in the droplets.

However, emulsions containing perfluorinated oil can suffer from variousproblems. For example, aqueous droplets are typically buoyant inperfluorinated oil, which can create problems during dropletmanipulation. The buoyant droplets may be more likely to be damaged byexposure to air above the emulsion, particularly when heated. Also, suchemulsions may require removal of excess oil below the droplets toposition the droplets closer to a heat source. Furthermore, the dropletsmay be difficult to stabilize for heat treatment, such as thermocyclingto promote amplification, and may be difficult to singulate prior todetection.

SUMMARY

The present disclosure provides a system, including methods andcompositions, for making and using emulsions that include a silicone oiland a silicone surfactant. The emulsions may include aqueous dropletsdisposed in a continuous phase that includes a silicone oil and asilicone surfactant. The aqueous droplets may contain an analyte,optionally at partial occupancy, and/or a luminescent (e.g.,photoluminescent) reporter. An assay of the analyte may be performedwith the droplets. In some cases, signals may be detected from thedroplets, and a characteristic of the analyte, such as an analyte levelor activity, may be determined based on the signals.

DETAILED DESCRIPTION

The present disclosure provides a system, including methods andcompositions, for making and using emulsions that include a silicone oiland a silicone surfactant. The emulsions may include aqueous dropletsdisposed in a continuous phase that includes a silicone oil and asilicone surfactant. The aqueous droplets may contain an analyte,optionally at partial occupancy, and/or a luminescent (e.g.,photoluminescent) reporter. An assay of the analyte may be performedwith the droplets. In some cases, signals may be detected from thedroplets, and a characteristic of the analyte, such as an analyte levelor activity, may be determined based on the signals.

A composition is provided. The composition may comprise a continuousphase that includes a silicone oil and a silicone surfactant, andaqueous droplets disposed in the continuous phase. The droplets mayinclude an analyte at partial occupancy.

A method of performing an assay is provided. In the method, an emulsionmay be formed that includes droplets disposed in a continuous phase. Thecontinuous phase may include a silicone oil and at least one siliconesurfactant. Data related to an analyte disposed in the disperseddroplets may be collected.

The system may have substantial advantages. The emulsion may prevent adroplet-air interface from forming because the droplets of the emulsionmay be denser than the oil, which causes the droplets to be positionedbelow any excess oil in a PCR tube or plate. The droplets may be stableto thermocycling during PCR without the need for a protein skin aroundeach droplet. Also, the droplets may sink, which allows the droplets tobe packed readily prior to detection or other manipulation, affordingmore control over droplet transport in a droplet reader (or other devicefor droplet manipulation), and potentially avoiding a need for afocusing fluid in a droplet reader (or other device for dropletmanipulation).

Further aspects of the present disclosure are presented in the followingsections: (I) oil phase, (II) aqueous phase, (III) detection of signalsfrom droplets, and (IV) examples.

I. Oil Phase

At least one silicone oil may be used to form the emulsion. The oil mayform at least a majority of the continuous phase of the emulsion. Theoil may have a low viscosity, such as less than about 10, 5, or 2centistokes, or about one or less centistoke. The oil may benon-volatile and/or may have a density less than water. A silicone oil,as used herein, includes one or more component compounds having apolysiloxane backbone with organic side chains. That is, the backbone ofa silicone oil includes a chain of alternating silicon and oxygen atoms(—Si—O—Si—O—Si—), where the silicon atoms are substituted by varioushydrocarbon moieties.

A variety of silicone oils, and blends of silicone oils, may be suitablefor use as a continuous phase for the purposes of the present invention.One exemplary silicone oil that may be a suitable continuous phase, or acomponent of a continuous phase, is a trisiloxane oil, such asoctamethyl trisiloxane. Alternatively, one or more components of thecontinuous phase may be a polydimethylsiloxane (PDMS) polymer. In onepreferred embodiment of the invention, the continuous phase includes aPDMS silicone oil having a relatively low kinematic viscosity (the ratioof dynamic viscosity to density) of about 0.1 to about 10 cSt, morepreferably about the silicone oil is a 5 cSt PDMS.

The stability of an aqueous-silicone oil emulsion may be enhanced by theaddition of a surfactant (amphiphile). However, some surfactants thatprovided good emulsion characteristics when used with carbon-based oilshave been found to provide less satisfactory results when used incombination with silicone oils. Silicone surfactants may be used tostabilize the aqueous-silicone oil emulsion without suffering from thedisadvantages of previously used surfactants.

At least one silicone surfactant may be included in the emulsion,generally as part of the oil phase. The surfactant may be heat stable,that is, stable to heating to a temperature of at least about 70° C.,80° C., 90° C., 100° C., or 110° C. for at least about 1, 2, 10, 30, 60,120, or 180 minutes, among others. The surfactant may be biocompatible,biologically inert, and/or compatible with one or more chemicalreactions, such as enzyme-catalyzed reactions. For example, the surfacemay be compatible with amplification (i.e., may not inhibitamplification substantially), such as by the polymerase chain reaction,in droplets of the emulsion. The surfactant collectively, or one or moresurfactants individually, may be present at any suitable concentrationin the continuous phase or in the oil before the emulsion is formed.Exemplary concentrations that may be suitable include about 0.1% to 10%,0.2% to 5%, 0.5% to 2%, or about 1% by weight, among others.

The silicone surfactant may be described by the following generalformula:[SILICONE BACKBONE][ALKYL]_(x)[POLYETHER]_(y)[POLYSILOXANE]_(z)where x is 0-5, y is 1-35, and z is 2-50.

In the above general formula, the SILICONE BACKBONE moiety correspondsto a polysiloxane chain having a repeating silicon-oxygen structuresubstituted by alkyl substituents, having the formula:

Here, a is 5-500, and each R moiety is hydrogen or an alkyl substituent,which may be the same or different, although each R moiety is typicallya lower alkyl group having 1-6 carbons. Preferably, the silicone chainis substantially completely substituted by methyl groups, and theresulting silicone chain is a polydimethylsiloxane (PDMS) polymer.

The silicone backbone moiety may be further substituted by one or moreadditional substituents, represented in the general formula by themoieties ALKYL, POLYETHER, and POLYSILOXANE. The particular nature andnumber of the additional substituents may be selected to tailor thehydrophobicity and/or hydrophilicity of the surfactant, and thus tofine-tune the utility of the surfactant for stabilizing a particularaqueous/silicone oil emulsion composition. For example, the siliconebackbone chain of the surfactant may be further substituted by one ormore hydrophilic moieties, where the hydrophilic moiety is selected toconfer enhanced hydrophilicity on the surfactant. The silicone backbonemay be substituted by 1-5 hydrophilic moieties, which may be the same ordifferent.

An example of an appropriate hydrophilic moiety is a POLYETHER moiety,such as an organic polyether chain. The organic polyether may be alinear alkyl that incorporates a plurality of bridging oxygen atoms. Forexample, the hydrophilic moiety may be a polyethylene glycol (PEG)moiety or a polypropylene glycol moiety. In one embodiment, thehydrophilic moiety includes a polyethylene glycol (PEG) moiety havingthe formula:

Here, b is 5-300, or more typically b is 15-100. In some embodiments,the hydrophilic moiety may be an organic polyether that is itselffurther substituted by an inorganic polysiloxane.

Where a hydrophilic moiety is a POLYSILOXANE moiety, it may include aninorganic polysiloxane that is optionally substituted one or more timesby alkyl having 1-carbons. In one example, the inorganic polysiloxaneside chain is a polydimethylsiloxane (PDMS) chain, such as, for example,a polysiloxane substituent having the formula:

Here, c is 2-100. Alternatively, the polysiloxane moiety may include abranched polysiloxane chain, such as a secondary polysiloxane having theformula:

Here, each c is independently 2-100. In yet another alternative, thepolysiloxane moiety may include a tertiary branched polysiloxane chain,such as, for example, a hydrophilic moiety having the formula:

Here, each c is independently 2-100. A polysiloxane substituent maytherefore be described by the formula:

Here, each R is hydrogen or alkyl having 1-6 carbons, d is 1-3, f is(3-d), and each c is independently 2-100. Each polysiloxane moiety maybe derivatized by one or more alkyl groups having 1-6 carbons, forexample, at the side chain terminus.

Alternatively or in addition, the silicone backbone chain may besubstituted by one or more hydrophobic moieties, where the hydrophobicmoiety is selected to confer enhanced hydrophobicity on the surfactant.The silicone backbone chain may be substituted by 1-5 hydrophobicmoieties, which may be the same or different. Typically, the hydrophobicmoiety, if present, is an ALKYL moiety, which includes a linear alkylside chain having 8-14 carbons.

The silicone surfactant may have any suitable structure, including acomb structure, a di-block structure, a tri-block structure, azwitterionic structure, an X block structure, a Y block structure, or acombination thereof, among others. In some cases, the siliconesurfactant may include the specific surfactants ABIL® EM 90 or ABIL® EM180, and/or one or more of Surfactants A-F shown below:

Surfactant A

Surfactant B

Surfactant C

wherein

each g is independently 5-200;

each h is independently 1-5;

each i is independently 1-5;

each j is independently 15-50; and

each k is independently 2-100.

Surfactant D

wherein

each g is independently 5-200;

each h is independently 1-5;

each i is independently 1-5;

each j is independently 15-50; and

each k is independently 2-100.

Surfactant E

wherein

each g is independently 5-200;

each h is independently 1-5;

each i is independently 1-5;

each j is independently 15-50; and

each k is independently 2-100.

Surfactant F

In one exemplary and preferred embodiment, the silicone surfactant mayhave the structure

wherein

each g is 10-25;

each h is independently 1-3;

each i is independently 2-4;

each j is independently 15-20; and

each k is independently 10-50.

In some cases, the emulsion, the continuous phase, and/or an oilcomposition used to prepare the emulsion, may include a silicone resinsolution, for example such as XIAMETER® RSN-0749, also known as DOWCORNING® 749 FLUID, and optionally at least one silicone surfactant asdescribed above. Alternatively, or in addition, the emulsion, continuousphase, and/or oil composition used to prepare the emulsion may include asilicone resin solution such as GP-422, available from GENESEE POLYMERSCORPORATION (Burton, Mich.). The silicone resin solution may be presentat any suitable concentration, such as a concentration of about 0.1% to10%, 0.2% to 5%, 0.5% to 2%, or about 1% by weight, among others.

In some cases, the emulsion, the continuous phase, and/or an oilcomposition used to prepare the emulsion, may includedecamethylcyclopentasiloxane, and/or octamethylcyclotetrasiloxane, whichmay be present at any suitable concentration, such as a concentration ofabout 0.0.5% to 5%, 0.1% to 2.5%, 0.25% to 1%, or about 0.5% by weight,among others.

In some cases, the emulsion, the continuous phase, and/or an oilcomposition used to prepare the emulsion, may includetrimethylsiloxysilicate (TSS). The TSS may be present at any suitableconcentration, such as a concentration of about 0.0.5% to 5%, 0.1% to2.5%, 0.25% to 1%, or about 0.5% by weight, among others.

A variety of silicone-based surfactants may be designed and prepared viathe modification of existing carbon-based surfactants through thesubstitution of one or more of the hydrocarbon backbone chains and/orpolyether side chains with a corresponding polysiloxane chain. Thedetails of the geometry and chain length of the polydimethylsiloxanebackbone and side chains could be readily tailored to meet theparticular demands of the particular emulsion system in which thesurfactant is used (or intended to be used).

Further aspects of making and using emulsions, and compositions thereof,are disclosed in the documents listed above under Cross-References,which are incorporated herein by reference.

II. Aqueous Phase

The emulsion may contain aqueous droplets formed from a continuousaqueous phase. The droplets may be monodisperse. The droplets mayinclude, and/or may be formed with an aqueous phase including, anaqueous surfactant. The droplets also may include at least one analyteto be characterized in a droplet-based assay. The analyte may be presentat partial occupancy, such that one or more (e.g., a plurality) of thedroplets contain no copies of the analyte, one or more (e.g., aplurality) of the droplets may contain a single copy (only one copy) ofthe analyte, and, optionally, yet one or more of the droplets (e.g., therest of the droplets) may contain two or more copies of the analyte. Theterm “partial occupancy” is not restricted to the case where there is nomore than one copy of a particular analyte in any droplet. Dropletscontaining an analyte at partial occupancy may, for example, contain anaverage of more than, or less than, about one copy, two copies, or threecopies, among others, of the analyte per droplet when the droplets areprovided or formed. Copies of an analyte may have a random distributionamong the droplets, which may be described as a Poisson distribution.Exemplary analytes include a biological material, such as a cell, aviral particle, an organelle, a nucleic acid target or template, aprotein, an amino acid, a lipid, a carbohydrate, a hormone, a receptor,a ligand, a metabolite, a catabolite, an ion, or the like. The analytemay be characterized in the droplets to determine any suitablecharacteristic, such as a qualitative level (present/absent), aquantitative level or concentration, an activity, or any combinationthereof, among others.

The aqueous droplets may include a reporter. The reporter may be orinclude a dye, which in turn may include a luminophore (e.g., afluorophore). The reporter may be configured to interact, associate, orbind to the analyte, a reaction product representing the analyte, or thelike. In any event, the reporter may be configured to report theoccurrence and/or extent of a reaction, such as a chemical reaction inthe droplets. Alternatively, or in addition, the reporter may report pH,a change in cellular ion concentration, cellular activity or death,protein function, viscosity, temperature, etc. Exemplary reporters fornucleic acid amplification include a dye-labeled probe, a genericreporter (e.g., an intercalating dye), or the like. In some cases, asignal may be detected by direct detection of droplets withoutinvolvement of a reporter.

The droplets may contain all necessary components to report acharacteristic or activity occurring in the droplets, or additionalmaterials may be delivered to the droplets during droplet use. In somecases, the aqueous droplets may contain a reaction mixture to perform achemical reaction in the droplets. The reaction may be anenzyme-catalyzed reaction, and the droplets may contain at least oneenzyme.

Further aspects of aqueous droplets and suitable contents of aqueousdroplets are disclosed in the documents listed above underCross-References, which are incorporated herein by reference.

III. Detection of Signals from Droplets

Signals may be detected from the droplets. The signals may be detectedwith the droplets moving or disposed in a one-dimensional array, atwo-dimensional array, or a three-dimensional array, among others. Eachsignal may represent an electrical characteristic of the droplets, athermal change, a pH, or detected light (e.g., luminescence (intensity,lifetime, polarization, energy transfer (e.g., FRET), etc.), absorbance,reflectivity, methods of direct detection, not involving reporters,etc.), among others. The signals may represent light detected from thedroplets, such as light emitted in response to irradiation withexcitation light. One or more signals may be detected from each of aplurality of the droplets. The signals may be detected from the dropletsserially, such as while the droplets flow past a detector, in parallel,such as by imaging the droplets or with parallel flow channels (e.g.,with a multi-channel singulator), laser scanning, or a combinationthereof, among others. The signals may be processed to quantitatively orqualitatively determine a characteristic of the analyte and/or thedroplets.

The term “luminescence” means emission of light that cannot beattributed merely to the temperature of the emitting body. Exemplaryforms of luminescence include photoluminescence, chemiluminescence,electroluminescence, or the like. A “luminophore” is any atom orassociated group of atoms capable of luminescence. Photoluminescence isany luminescence produced in response to irradiation with excitationlight and includes fluorescence, phosphorescence, etc. Accordingly, aluminophore may be a fluorophore or a phosphor, among others.

Further aspects of signal detection from droplets, signal processing,and assays that can be performed with emulsions are disclosed in thedocuments listed above under Cross-References, which are incorporatedherein by reference.

IV. Examples

The following examples, presented as a series of numbered paragraphs,describe selected aspects of silicone surfactants, emulsions containingsilicone surfactants, and methods of using such emulsions indroplet-based assays. These examples are intended for illustration onlyand should not limit the entire scope of the present disclosure.

1. A silicone surfactant having the formula:[SILICONE BACKBONE][ALKYL]_(x)[POLYETHER]_(y)[POLYSILOXANE]_(z)where x is 0-5, y is 1-35, and z is 2-50.

2. The silicone surfactant of paragraph 1, wherein SILICONE BACKBONE hasthe formula:

wherein a is 5-500, and each R moiety is independently hydrogen or analkyl having 1-6 carbons.

3. The silicone surfactant of paragraph 1, wherein ALKYL is an alkylmoiety having 8-14 carbons.

4. The silicone surfactant of paragraph 1, wherein POLYETHER is anorganic polyether side chain.

5. The silicone surfactant of paragraph 4, wherein POLYETHER has theformula

wherein b is 5-300.

6. The silicone surfactant of paragraph 1, wherein POLYSILOXANE is aninorganic polysiloxane moiety.

7. The silicone surfactant of paragraph 6, wherein POLYSILOXANE is apolydimethylsiloxane side chain.

8. The silicone surfactant of paragraph 6, wherein POLYSILOXANE has theformula:

wherein each R is independently hydrogen or alkyl having 1-6 carbons,each c is independently 5-100, d is 1-3, and f is (3-d).

9. The silicone surfactant of paragraph 1, having the formula

wherein

each g is independently 5-200;

each h is independently 1-5;

each i is independently 1-5;

each j is independently 15-50; and

each k is independently 2-100.

10. The silicone surfactant of paragraph 1, having the formula

wherein

each g is independently 5-200;

each h is independently 1-5;

each i is independently 1-5;

each j is independently 15-50; and

each k is independently 2-100.

11. The silicone surfactant of paragraph 1, having the formula

wherein

each g is independently 5-200;

each h is independently 1-5;

each i is independently 1-5;

each j is independently 15-50; and

each k is independently 2-100.

12. A composition comprising at least one silicone surfactant accordingto one of paragraphs 1-11.

13. The composition of paragraph 12, wherein the composition is anemulsion.

14. The composition of paragraph 12 or 13, wherein the compositionincludes a silicone oil.

15. The composition of paragraph 14, wherein the silicone oil includesone or more of octamethyl trisiloxane and a 5 cSt PDMS.

16. The composition of any one of paragraphs 12-15, wherein the siliconesurfactant is present in the composition at a concentration of 0.1% to10% in an oil phase.

17. A composition comprising: (A) a continuous phase including asilicone oil and a silicone surfactant; and (B) aqueous dropletsdisposed in the continuous phase, the droplets including an analyte atpartial occupancy.

18. The composition of paragraph 17, wherein the silicone surfactant isa silicone surfactant according to one of paragraphs 1-11.

19. The composition of paragraph 17, wherein the aqueous dropletsinclude a photoluminescent reporter.

20. The composition of one of paragraphs 17-19, wherein the analyte is anucleic acid.

21. A method of performing an assay, comprising: (A) forming an emulsionincluding droplets disposed in a continuous phase that includes asilicone oil and at least one silicone surfactant according to one ofparagraphs 1-11; and (B) collecting data related to an analyte disposedin the droplets.

22. The method of paragraph 21, wherein the analyte is a biomaterial.

23. The method of paragraph 21, wherein the analyte is a nucleic acid.

24. The method of any one of paragraphs 21 to 23, wherein copies of theanalyte are present in the droplets at partial occupancy when theemulsion is formed.

25. The method of any one of paragraphs 21 to 24, wherein the siliconeoil includes a polydimethylsiloxane.

26. The method of paragraph 25, wherein the polydimethylsiloxane is a 5cSt polydimethylsiloxane.

27. The method of any one of paragraphs 21 to 26, wherein the continuousphase includes octamethylcyclotetrasiloxane.

28. The method of paragraph 27, wherein the octamethylcyclotetrasiloxaneis present at a concentration of about 0.1% to 2% by weight or about0.5% by weight.

29. The method of any one of paragraphs 21 to 28, wherein the continuousphase includes a trimethylsiloxysilicate.

30. The method of paragraph 29, wherein the trimethyloxysiloxysilicateis present at a concentration of about 0.1% to 5% by weight.

31. The method of any one of paragraphs 21 to 30, wherein the siliconesurfactant includes a silicone surfactant having a comb structure.

32. The method of any one of paragraphs 21 to 30, wherein the siliconesurfactant includes a silicone surfactant having a di-block or tri-blockstructure.

33. The method of any of paragraphs 21 to 32, wherein the siliconesurfactant is present at a concentration of about 0.2% to 5% by weight.

34. The method of paragraph 33, wherein the silicone surfactant ispresent at a concentration of about 0.5% to 2% by weight.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.Further, ordinal indicators, such as first, second, or third, foridentified elements are used to distinguish between the elements, and donot indicate a particular position or order of such elements, unlessotherwise specifically stated.

We claim:
 1. A method of performing an assay, the method comprising: (A)forming an emulsion including aqueous droplets dispersed in a continuousphase, the aqueous droplets containing an analyte, the continuous phaseincluding a silicone oil and a silicone surfactant, the siliconesurfactant having the formula

the formula

or the formula

wherein each g is independently 5-200; each h is independently 1-5; eachi is independently 1-5; each j is independently 15-50; each k isindependently 2-100; and (B) detecting a signal related to the analytefrom the aqueous droplets.
 2. The method of claim 1, wherein only asubset of the aqueous droplets contains the analyte.
 3. The method ofclaim 1, wherein the analyte is a nucleic acid.
 4. The method of claim1, wherein the step of detecting a signal includes a step of detectinglight.
 5. The method of claim 4, wherein the aqueous droplets contain aluminescent reporter, and wherein the step of detecting light includes astep of detecting light from the luminescent reporter.
 6. The method ofclaim 5, wherein the luminescent reporter includes a dye-labeled probe.7. The method of claim 5, wherein the luminescent reporter includes anintercalating dye.
 8. The method of claim 1, further comprising a stepof performing an enzyme-catalyzed reaction in the aqueous droplets. 9.The method of claim 1, wherein the silicone oil includes octamethyltrisiloxane, cyclopentasiloxane, or a trimethylsiloxysilicate at aconcentration of about 0.1% to 5%.
 10. The method of claim 1, whereinthe silicone oil includes one or more of octamethyl trisiloxane andpolydimethylsiloxane.
 11. The method of claim 1, wherein the siliconesurfactant is present in the continuous phase at a concentration ofabout 0.1% to 10% by weight.